C. difficile colitis is a toxin-mediated disease, which is dependent on the actions of Toxin A (TcdA) and/or Toxin B (TcdB). Although the molecular mechanisms by which TcdA and TcdB modify Rho-subfamily GTPases are well defined, the pathways connecting those events to colitis and inflammatory diarrhea remain elusive. In this sub-project of MARCE Research Program V (Interactions of Select Agent Toxins and Toxins of Emerging Bacterial Pathogens with Host Cells), we will combine the analytical methods of systems biology with expression profiles (gene expression and protein modification) and associated biological consequences, in order to elucidate the pathways affected by the specific toxin-mediated glucosylation of one or more small GTPases. Cultured human cells alone and in combinations (intestinal epithelium and other cell types directly affected by the toxins) will be studied in vitro and mouse ligated ileal loops will be studied in vivo to characterize the phenotypes induced by the toxins and to determine the optimal conditions for collection of the gene array data. A novel pathway-based compendium analysis will be used to overlay the in vitro and in vivo transcriptional profiles on a literature-derived pathway knowledge database, thereby identifying signaling networks altered by the toxins (SA 1). Concurrently, data derived through gene expression arrays will be validated using qRT-PCR, Western blotting, phospho-protein analyses and in situ hybridization. Partial least-squares regression analysis will be applied to the collective data, in order to characterize the key signaling metrics for the phenotypes elicited by TcdA and TcdB (SA 2). New hypotheses will then be generated and tested by making biological and biochemical manipulations of the newly recognized pathways and signaling molecules and determining the biological consequences with and without the toxins (SA 3). This exciting project reflects a close interaction of three groups, representing: 1) microbial toxin action and molecular pathogenesis (Hewlett); 2) pathogenesis of diseases caused by enteric pathogens and enterotoxins (Guerrant and Warren) and 3) systems bioengineering in infectious diseases (Papin).